The present invention relates generally to methods and materials useful in development of immunological responses protective against malarial infection in susceptible vertebrates, including humans.
Human malaria is caused by species of parasitic organisms of the genus Plasmodium. It is transmitted by mosquitoes which ingest sexual forms of the parasite in blood meals. Sporozoite forms of the parasite develop in the mosquito and are transmitted to new host individuals bitten by the insect. In the new host, the sporozoite parasites develop and multiply in an "exoerythrocytic" cycle in the liver without inducing clinical symptoms. The merozoite forms resulting from exoerythrocytic propagation then invade host erythrocytes, initiating an "erythrocytic" cycle of development and prompting the clinical symptoms of malaria. Destruction of red blood cells occurs on a 48-hour cycle with P.vivax, P.ovale and P.falciparum, and every 72 hours with P.malariae. Characteristic chills-fever-sweat malarial symptoms follow this cyclic pattern, being induced by rupture of infected red blood cells by the mature asexual forms (schizonts), releasing merozoites that quickly invade new red cells. In contrast to the exoerythrocytic stage, erythrocytic merozoites induce an array of humoral responses in the host, as demonstrated by appearance of blood serum antibodies detectable by complement fixation, precipitation, agglutination, and fluorescent antibody tests.
Relapse results from periodic release of infective merozoites from the liver. When the erythrocytic cellular and humoral protection against the erythrocytic phase of the disease is deficient or reduced by concurrent infection, age, trauma, or other debilitating factors, relapse of clinical malaria occurs until the erythrocytic cycle is again controlled by a humoral and thymus-dependent cell-mediated host response. True relapse, as opposed to a delayed exoerythrocytic cycle or a recrudescence of erthrocytic infection, generally will occur for up to 5 years with P.vivax and possibly 2-3 years for P.ovale. P.malariae appears to recur only as a recrudescent erythrocytic infection, sometimes lasting 30 or more years after the primary infection. P.falciparum may have a short-term recrudescence and also does not develop a true relapse from liver-developed merozoites because only one exoerythrocytic liver phase develops during P.falciparum infection.
Information concerning immunological resistance to malarial infection has been developed from a variety of sources over the years. See, e.g., Chapter 41 in "Basic & Clinical Immunology", 3rd Ed., Fudenberg, et al., eds. [Lange Medical Publications, Los Altos, Calif., (1980)]. Much of this information is based on African populations wherein the disease state is endemic. It has been determined, for example, that a very gradual long-term resistance to falciparum malaria is acquired in African populations. The resistance develops years after the onset of severe disease among nearly all children over 3 months of age. (Initial passive protection is present owing to transplacental maternal IgG.) There are estimates of a million malaria deaths a year in Africa, chiefly among children under five. Even after surviving childhood infection, a large proportion of adults nonetheless remain susceptible to infection and show periodic parasitemia, even though their serum contains "protective" antiplasmodial antibodies. In hyperendemic areas of Africa, it is believed that nearly all residents harbor a continuous series of falciparum infections of low to moderate pathogenicity throughout their lives. The immune response that leads to protection is thought to be the production of complement-independent antibody that inhibits entry of merozoites into the host erythrocytes. All immunoglobulin classes are elevated in the serum of malaria patients, but IgG levels appear to correlate best with the degree of malaria protection (or control of acute manifestations).
Chemical (drug) treatment of clinical symptoms rather than immunization has been the major focus of malaria research for decades. A first major approach to development of anti-malarial vaccines has involved attempts to induce protective immunity using sporozoites inactivated by, e.g., ultraviolet light, formalin or mechanical disruption. These agents reputedly induce a short-term, thymus-dependent, species- and strain-specific immunity active only against the exoerythrocytic, sporozoite infection. This approach has generally involved use of sporozoites dissected from irradiated mosquitoes or by inoculation through the bite of irradiated mosquitoes. Only mature infective sporozoites have been found to be immunogenic and adjuvants appear to be unnecessary. This method is limited by the difficulty in storing the vaccine; by inability to culture and therefore obtain large amounts of immunizing antigen; by the requirement of intravenous administration of the vaccine; and by the continuing susceptibility of the immunized person to a merozoite infection (should even a single sporozoite succeed in developing in the liver).
Recent attempts have been made to bring genetic engineering manipulative techniques to bear on development of specific proteinaceous isolates which might possess the protective antigenic capability of the entire sporozoite fragments. Success in these endeavors may result in alleviation of the generation, storage and delivery problems noted above. It will remain the case, however, that if a single sporozoite (from among hundreds injected by a single mosquito bite) survives the host's vaccine-induced immune response, a severe erythrocytic stage infection can ensue. See, Marshall, Science, 219, pp. 466-467 (1983).
A second general approach to immunization has involved use of killed or inactivated merozoite vaccines. See, generally, Cohen, Proc.Royal.Soc.London, 203, pp. 323-345 (1979). Research efforts in this area have been aided greatly by the procedures developed by Trager and Jensen [Science, 193, pp. 673-675 (1976)] relating to continuous culture methods for in vitro propagation of erythrocytic stages of parasites.
Merozoite vaccines are believed to induce formation of multiple antibodies, some of which react with red cell surfaces and selectively agglutinate infected cells, generally producing a strain- and species-specific alleviation of clinical symptoms. New infections can still develop, since there is no protection against sporozoites or the exoerythrocytic cycle. So long as the humoral antibody titer is high, however, merozoites (but not gametocytes) will be destroyed, and symptoms will generally not develop. Rhesus monkeys vaccinated with P.knowlesi merozoites (normally quickly killed by this form of malaria) have been reported to be fully protected for 18 months.
Freund's Complete Adjuvant (FCA) or synthetic adjuvants are required for merozoite antigen use and thus constitutes a major deterrent to development of a human vaccine. More recent studies using karyotype-selected Aotus monkeys infected with human P.falciparum, reported prolongation of life in owl monkeys vaccinated with parasite material cultivated in vitro when the synthetic adjuvant muramyl dipeptide was used instead of FCA. In the rhesus monkey immunization studies, helper T cells, other cell-mediated effector mechanisms, and humoral antibody all appear to be involved. Extracellular merozoites are specifically inhibited by IgG and IgM in the absence of complement. Immunization in Rhesus monkeys reportedly induces complete elimination of parasites after 1-3 weeks, whereas natural immunity following repeated infection and drug cure is associated with chronic relapsing parasitemia. Immunization probably is associated with far fewer soluble circulating antigens than natural infection, which preferentially stimulates suppressor cells or lymphocyte mitogens, all of which favor parasite survival. Among the difficulties associated with immunization with merozoites are risks of contamination of the merozoite vaccine with blood group substances acquired during its cultivation (inducing anemia) and substantial potential problems of vaccine delivery, cost, and acceptance.
Among the most recent reports of work relating to merozoite vaccines is that of McColm, et al., Parasite Immunology, 4, pp. 337-345 (1982). This publication followed the extensive prior report of Mitchell, et al., Bull. W.H.O., 57, (Supp. 1), pp. 189-197 (1979) and of Desowitz, Experimental Parasitology, 38, pp. 6-13 (1975) in the ongoing study of the effects of various adjuvants on merozoite vaccine efficacy.
Apart from work directed to development of whole, killed or inactivated, merozoite vaccines, investigations spanning the last four decades have had as their focus the immunological properties of host and parasite antigens associated with the entirety of the erythrocytic stage of malarial parasite development. For example, antigenic proteins of parasite origin were detected in the plasma or serum of monkeys, ducks. rodents, chickens, and man with acute malaria as early as 1939. Partial protection against challenge infection was demonstrated in chickens and monkeys with blood plasma derived antigens of Plasmodium gallinaceum and Plasmodium knowlesi, respectively [Todorovic, et al., Ann.Trop.Med.Parasitol., 61, pp. 117-124 (1967); Collins, et al., Am.J.Trop.Med. & Hyg., pp. 373-376 (1977)]. Todorovic and his associates [Am.J.Trop.Med. & Hyg., 17, pp. 685-694 (1968); Am.J.Trop.Med. & Hyg., 17, pp. 695-701 (1968); and Trans.R.Soc.Trop.Med.Hyg., 61, pp. 51-57 (1968)], demonstrated that fluorescein-conjugated antibody specific for soluble P.gallinaceum serum antigens reacted with free merozoites and was capable of activating macrophages. The antigens were labile to temperatures greater than 65.degree. C., sensitive to proteolytic enzymes and contained a lipid component. Additionally, fluorescein-conjugated antibody prepared to the soluble antigens reacted with both infected erythrocyte cytoplasm and the parasite if the erythrocytes contained mature parasite forms. However, in erythrocytes containing immature ring forms, only the parasite was stained. Subsequent studies by these workers suggested that temperature, enzymatic degradation and antigen-antibody complexes occurring in the plasma of affected animals were among the elements which degraded the immunogenicity of these antigens and minimized their usefulness as vaccines.
McGregor, et al., [Lancet, 1, pp. 881-884 (1968)]; Wilson, et al., [Lancet, 2, pp. 201-205 (1969)]; McGregor, et al., [Trans.R.Soc.Trop.Med.Hyg., 65, pp. 136-151 (1971)]; and Williams, et al., [Af.J.Med.Sci., 4, pp. 295-307 (1972)], relate to demonstrations of the presence of soluble antigens in the plasma of human beings infected with an African strain of P.falciparum. Characterization of the majority of the soluble antigens found in the serum showed them to be heat stable at 100.degree. C. [Wilson, et al., Immunology, 3, pp 385-398 (1973)]. Consequently, they were called "S" antigens. Molecular weights reported for S antigens ranged from 60,000 to 210,000 daltons. Groups of soluble plasmodial antigens not usually found in the serum ("La", "Lb", and "R" antigens), had properties different from S antigens. L antigens were reportedly more immunogenic than S antigens and rapidly reacted with antibody leading to soluble antigen-antibody complexes in the serum [Wilson, et al., Lancet, 2, pp. 201-205 (1969); Houba, et al., Af.J.Med.Sci., 4, pp. 309-317 (1972); and Wilson, et al., Immunology, 3, pp. 385-398 (1973)]. Saul, et al., [Tropenmed. Parasitol., 28, 302-318 (1977)] demonstrated that a soluble protein-containing immunogen could be obtained by washing sonically freed P.berghei parasites with cold saline. Further work by Kreier's group [Grothaus, et al., Infect. and Immunol., 1, pp. 245-253 (1980)], is reported to show that the soluble material was more immunogenic than the intact parasites.
The occurrence of Plasmodium-associated antigens in infected plasma suggested that such antigens may be released from the parasitized erythrocytes. Membranes of erythrocytes parasitized with P.knowlesi that were thus subjected to immunochemical analysis have been shown to contain several proteins of parasite origin in the molecular weight range of 50,000 to 65,000 daltons [Wallach, et al., J.Mol.Med., 2, pp. 119-136, (1977) ; and Deans, et al., Parasitology, 77, pp. 333-344 (1978).
The most recently reported developments in the proposed use of antigenic fragments associated with erythrocytic stages of malarial parasite growth have had their origins at the Wellcome Foundation in the United Kingdom. More specifically, U.K. published Patent Application Ser. Nos. 2,096,893 and 2,099,300 both report that, prior to the development described, "Attempts have been made to define the diversity of protein antigens associated with merozoites. However, no specific antigens capable of inducing a protective response by the host or specifically recognized by such a protective response have been isolated and characterized." Both published applications are said to relate to "protection inducing antigens of parasites of the genus Plasmodium" and both describe the use of affinity separations (involving monoclonal antibodies) to isolate merozoite and schizont form antigens.
As specific examples of practice of the development, both published British applications describe isolation of antigens associated with murine-specific malarial species, Plasmodium yoelii. Briefly summarized, erythrocytes from infected cells of mice are lysed, centrifuged and solubilized with a variety of detergents to yield a supernatant containing erythrocyte soluble proteins, some erythrocyte membrane proteins and an estimated "70% of the parasite antigens". The solubilized material is then passed through an immunoabsorbant column to which specific monoclonal antibodies were bound. The eluate of the antigen/antibody absorption is concentrated and dialyzed to yield non-glycosylated antigens having a molecular weight of 2.35.times.10.sup.5 or 1.95.times.10.sup.5 (assertedly corresponding to merozoite- and schizont-associated antigens). The antigenic isolates are reported to have been successfully used with Freund's Complete Adjuvant to protect mice against lethal challenge P.yoelii parasites. The applications go on to discuss similar attempts to isolate one or more antigens or antigenic fragments from Plasmodium falciparum parasitized erythrocytes, using a correspondingly specific monoclonal antibody. The resulting antigens were tested in vitro for cross-reactivity with P.yoelii antigen but not employed in any in vivo (antibody generation or infectious challenge) work.
Assuming that the projected isolations of P.falciparum schizont and merozoite antigens according to the procedures of U.K. published Patent Application Nos. 2,096,893 and 2,099,300 are as fruitful as the work reported for P.yoelli antigens, it is possible that the solubilized protein isolates may provide useful components for a human vaccine composition. Large scale production of antigenic materials, however, is likely to involve numerous difficulties, including problems in securing large quantities of human blood cells infected with late stages of parasites in large scale solubilization processing of erythrocytes free of red blood cell components, and in large scale maintenance and operation of antibody columns for affinity purification.
As previously noted, development of methods for continuous in vitro propagation of malarial parasites by Trager and Jensen, supra, has markedly assisted in the development of merozoite vaccines and the general study of erythrocytic malarial parasite stages. In a sense, it has also provided a means for detection and isolation of soluble antigens unaffected by the host's metabolic and immune systems. As an example of this type of research, most investigators found maximal quantities of protein material to accumulate in the culture medium during late schizogony and merozoite reinvasion. The possibility of the presence of Plasmodium-associated material in culture supernatant had been reported in cultures of P.knowlesi (Cohen, et al., 1969), P.falciparum (Wilson, 1974; Wilson and Bartholomew, 1975), and P.berghei, (Weissberger, et al., 1979). Wilson and Bartholomew (1975) detected antigens that were heat stable, partially heat labile and heat resistant, termed S, L and R antigens, respectively. Jepson, et al., Acta.Path.Microbiol.Scand., Sect. C, 89, 99103 (1981) reported the isolation of two distinct antigens of the S and R classes from the culture medium of growth of P.falciparum in human erythrocytes. The isolation procedure involved immunoabsorbant techniques and is said to have yielded approximately 3 milligrams of the two antigens from 800 milliliters of culture medium. The results were said to "show promise for further attempts to isolate other antigens from the culture medium, and for obtaining knowledge about the chemistry and biology of the isolated antigens". Similarly, Thelu, et al., WHO Bulletin, 60, pp. 761-766 (1982) reports on the chromatographic isolation of an "Antigen E" from cultured P.falciparum and correlations between this substance and antigens in sera of human patients in endemic areas.
Of interest to the background of the invention is research generally involving use of immunological adjuvants and especially pertinent are those publications which discuss adjuvants believed to be suited for incorporation into malaria vaccines. Sometimes referred to as "immunopotentiators", adjuvants are ordinarily defined as substances which operate to increase the rate at which an immune response develops, or increase the intensity of the response, or prolong the response, or simply to allow for the development of any response at all to an otherwise essentially non-immunogenic substance. Adjuvants are commonly categorized as either general potentiators of both cellular and immune responses or specific potentiators of responses to only certain antigens. See generally, Chapter 24 of "Basic & Clinical Immunology", supra.
It has consistently been the case of the nonsporozoite materials displaying potential as anti-malaria vaccine components are so weakly immunogenic as to absolutely require the use of oil and water adjuvants such as Freund's Complete Adjuvant (FCA) to develop any effect. Such adjuvants are not accepted for use in humans. In anticipation of the discovery of truly protective anti-malarial antigens, substantial and relatively continuous efforts have been made in the screening of existing adjuvants and the development of new adjuvants for vaccine use. U.S. Pat. No. 3.849,551, for example, proposes the use of Mycobacteria bovis, strain Calmette-Guerin bacillus (BCG) as an adjuvant for malaria vaccines, and Schenkel, et al. [J.Parasitol., 61, pp. 549-550 (1975)] propose mixtures of BCG with Adjuvant 65 as providing even more beneficial results. Desowitz [Experimental Parasitology, 38, pp. 6-13 (1975)] provided a comprehensive screening study of various adjuvants used with a P.berghei, blood-derived soluble antigens. Among the many results of tne study was the conclusion that ferric alum and aluminum chloride precipitated antigens were non-immunogenic while aluminum-alum-precipitated antigens might be protective. As previously noted, Mitchell, et al., supra, studied adjuvant effects for merozoite antigens and concluded that muramyldipeptide in mineral oil was partially effective in some studies and saponin was demonstrably effective in others. Siddiqui, et al. [Nature, 289, pp. 64-66 (1981)] reports on "effective immunization of monkeys with killed parasites and N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl-propanediamine) McColm, et al., supra, reported the testing of numerous adjuvants with a killed parasite vaccine and concluded that none were as effective as saponin, although FCA, aluminum hydroxide and C.parvum augmented immunity considerably. Correspondingly, the aforementioned U.K. published Patent Application Nos. 2,096,893 and 2,099,300 report use of FCA in vaccination tests designed to illustrate potential utility for the isolated antigen, but note that "convenient" adjuvants for use in vaccines include saponin, C.parvum and aluminum hydroxide.
The above remarks with respect to the background of the present invention establish that, despite decades of costly investigation and determined effort by countless investigators, there continues to exist a need in the art for readily available materials demonstrably useful as protective immunogens in antimalarial vaccines.